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Published ahead of print on September 10, 2004, doi:10.1164/rccm.200402-200OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 1331-1339, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200402-200OC

Original Article

Azithromycin Blocks Neutrophil Recruitment in Pseudomonas Endobronchial Infection

Wan C. Tsai, Michael L. Rodriguez, Katherine S. Young, Jane C. Deng, Victor J. Thannickal, Kazuhiro Tateda, Marc B. Hershenson and Theodore J. Standiford

Departments of Pediatrics and Medicine, Divisions of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, Michigan; and Department of Microbiology, Toho University School of Medicine, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Wan C. Tsai, M.D., University of Michigan Medical Center, Division of Pediatric Pulmonary Medicine, 6301 MSRB III, Box 0642, Ann Arbor, MI 48109–0642. E-mail: wctsai{at}med.umich.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrolides exert their effects on the host by modulation of immune responses. In this study, we assessed the therapeutic efficacy of azithromycin in a murine model of mucoid Pseudomonas aeruginosa endobronchial infection. The clearance of Pseudomonas from the airway of mice treated with the macrolide azithromycin was not different than untreated mice challenged with Pseudomonas beads. However, the azithromycin-treated mice showed a remarkable reduction in lung cellular infiltrate in response to Pseudomonas beads, as compared with untreated mice. This effect was associated with significant decreases in lung levels of tumor necrosis factor-{alpha} and keratinocyte-derived chemokine in azithromycin-treated mice compared with untreated mice. Furthermore, there was a significant reduction in the response of both mouse and human neutrophils to chemokine-dependent and -independent chemoattractants when studied in vitro. Inhibition of chemotaxis correlated with azithromycin-mediated inhibition of extracellular signal–regulated kinase-1 and -2 activation. This study indicates that the azithromycin treatment in vivo results in significant reduction in airway-specific inflammation, which occurs in part by inhibition of neutrophil recruitment to the lung through reduction in proinflammatory cytokine expression and inhibition of neutrophil migration via the extracellular signal–regulated kinase-1 and -2 signal transduction pathway.

Key Words: airway inflammation • chemotaxis • macrolides • neutrophils


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Persistent endobronchial infection due to Pseudomonas aeruginosa is a common and devastating complication of airway diseases such as cystic fibrosis (CF). Persistent airway colonization of CF airways by Pseudomonas has been implicated as an important stimulus in the development of lung disease, characterized by chronic unopposed neutrophil-dominated airway inflammation, progressive bronchiectasis, which results in high mortality (1–3). No intervention has been successful at eradicating P. aeruginosa from the airways of susceptible patients.

Azithromycin is an erythromycin-derived 15-membered ring azalide that is structurally modified to permit unusually enhanced intracellular accumulation, resulting in greater tissue penetration, extended elimination half-life, and extensive intracellular and extracellular antimicrobial activity (4–9). Azithromycin accumulates in higher concentration in host phagocytes than in extracellular compartment and has been shown to improve host phagocyte bactericidal activity synergistically in susceptible microorganisms (9). Its effect on inflammation is less well studied compared with those of 14-membered ring macrolides such as erythromycin or clarithromycin.

Multiple lines of evidence suggest that macrolide antibiotics exert potent immunomodulatory effects in the setting of infection, effects that may occur independent of direct bactericidal activity. Clinical observations in patients with diffuse pan-bronchiolitis and cystic fibrosis, chronic lung diseases similarly characterized by persistent endobronchial Pseudomonas infection, and airway neutrophilia indicate improvements in clinical outcomes with long-term macrolide therapy (4, 10–16). Erythromycin treatment in patients with diffuse pan-bronchiolitis has been associated with improved survival and reduced airway inflammation, as manifest by decreases in bronchoalveolar lavage fluid neutrophilia and levels of proinflammatory mediators (interleukin-8/cysteine-X-cysteine 8 [IL-8/CXCL8], IL-1ß, and leukotriene B4) (17, 18). In animal models of gram-positive and gram-negative bacterial pneumonia, in vivo treatment with erythromycin resulted in a dose-dependent attenuation of neutrophil recruitment to the lungs of mice challenged with aerosol inhalation of radiolabeled Staphylococcus aureus and Proteus mirabilis (19). Few studies have investigated immunomodulatory effects of macrolides in in vivo models of respiratory tract infection caused by Pseudomonas aeruginosa. Mice chronically intubated with a plastic tube precoated with P. aeruginosa in the bronchus were shown to have normalization of bronchoalveolar lavage fluid lymphocyte count and elevated CD4+/CD8+ ratio after oral therapy with clarithromycin (20). The same investigators subsequently noted a reduction in lung levels of IL-1ß, IL-2, IL-4, IL-5, interferon-{gamma} (IFN-{gamma}), and tumor necrosis factor-{alpha} (TNF-{alpha}) after clarithromycin treatment (21).

Available studies have provided conflicting results regarding the direct effects of macrolides on the host leukocyte function (22). In vitro observations suggest that erythromycin derivatives affect cellular immune responses by altering neutrophil oxidant production (19, 23–28), neutrophil degranulation (29, 30), and influencing phagocyte chemotaxis (19, 31). Macrolides might also influence recruitment of inflammatory cells by reducing the expression of chemotactic and activating cytokines (17, 32). Taken together, macrolides appear to target multiple components of the inflammatory cascade (31, 33). In this study, we investigated the effect of subcutaneous administration of azithromycin on the neutrophil-dependent innate immune response in an in vivo model of chronic Pseudomonas endobronchial infection and have defined mechanisms that may contribute to the immunomodulatory effects observed. Some of the results of these studies have been previously reported in the form of an abstract (34).


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Specific methods are provided in detail in the online supplement.

Reagents
Cytokine antibodies used in ELISAs, N-formyl-met-leu-phe (FMLP), Brewer Thioglycollate medium, IL-8, keratinocyte-derived chemokine (KC), primary antiphospho p42/44 mitogen-activated protein kinase (MAPK), goat secondary antibodies for Western blots, inhibitors of p38 MAPK (SB203580), p42/44 MAPK (U0126), and phosphatidylinositol 3-kinase (PI3K) (LY294002) were purchased.

Animals
C57BL/6J adult mice were purchased from The Jackson Laboratory (Bar Harbor, Maine) and housed in specific pathogen–free conditions.

Preparation and Administration of Pseudomonas Agar Beads
A mucoid P. aeruginosa strain (mPa5) isolated from an adult patient with cystic fibrosis was used in our studies. A modified agar bead method was employed (35).

Azithromycin Dosage Regimen
A commercially available intravenous formulation of azithromycin lactobionate (Zithromax IV; Pfizer, Inc., New York, NY) was used. Animals were subcutaneously administered 20 mg/kg of azithromycin or saline (untreated control) once daily for 3 days at the same time as intratracheal bacterial challenge as previously described (36). The specific pharmacokinetics of azithromycin dose, subcutaneous administration in C57BL/6J mice, and rationale for exposure duration in vitro are further detailed in the online supplement (9).

Determination of Lung P. Aeruginosa cfu
Lungs were removed aseptically and homogenized under a vented hood. Ten microliters of each serial 1:10 dilution was plated on 4% soy-base blood agar plates (Difco, Detroit, MI), and colonies were counted after 24 hours incubation at 37°C.

Cytokine ELISAs Assays
Murine TNF-{alpha}, IFN-{gamma}, IL-12, macrophage inflammatory protein-2 (MIP-2), lipopolysaccharide-induced CXC chemokine, and KC were quantitated using a modification of a double ligand method as previously described (37). Lung myeloperoxidase activity was quantified by a method as described previously (38).

Lung Inflammatory Cells Enumeration
Lungs were harvested from euthanized mice, suspended in penicillin/streptomycin-containing RPMI, type I collagenase, and DNase (both from Worthington, Freehold, NJ), and minced as previously described (38). Cells were counted in a hemocytometer, and cell differentials were determined by Wright-Giesma staining of cytospins with Diff Quik (Dade Behring, Newark, DE).

Isolation of Mouse and Human Neutrophils
Mice were injected intraperitoneally with 3 ml of sterile 3% Brewer Thioglycollate medium (39). Cells were harvested 5 hours later by peritoneal lavage. This procedure routinely yielded 50-million total cells when pooled, with 97% of the cells as neutrophils. Neutrophils from human peripheral blood were isolated on a Ficoll-Hypaque gradient and Dextran sedimentation. This method reproducibly yielded 12-million cells per milliliter, with greater than 98% neutrophils isolated. Neutrophils were suspended in Ca2+ and Mg2+ free Hanks' balanced salt solution and preincubated with 4 µg/ml azithromycin for 4 hours.

Chemotaxis Assay
Neutrophil chemotaxis was performed using a 12-well Boyden chemotaxis chamber (Neuroprobe, Cabin John, MD) as described (40). Samples were tested in quadruplicates. Chemotaxis was expressed as the number of migrated neutrophils per high-powered field magnified at x1,000.

Statistical Analysis
All data were compared using an unpaired two-tail (nonparametric) Mann-Whitney test or one-way analysis of variance with Bonferroni post-test by means of GraphPad Prism software, version 3.0 for Windows (San Diego, CA); p values were considered statistically significant if they were less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mice Challenged with Agar Beads Impregnated with Mucoid P. aeruginosa Develop Persistent Endobronchial Infection
To create a model of chronic nonlethal endobronchial infection that resembles the clinical setting in which humans develop Pseudomonas airway disease, we intratracheally challenged C57BL/6J mice with 5 x 105 cfu of mucoid P. aeruginosa (mPa5) impregnated in agar beads. The beads interfere with elimination of the bacteria leading to chronic infection. Mice administered sterile beads had no mortality, sterile lungs and a lung inflammatory profile similar to normal untreated animals (Figure 1B) (data not shown). In contrast, animals challenged with mPa-impregnated beads developed persistent Pseudomonas colonization, as P. aeruginosa could be recovered from the lungs greater than 12 days after challenge (Figure 1A). Mice challenged with mPa also demonstrated a substantial increase in the influx of neutrophils into the lungs, as determined by myeloperoxidase levels, a marker of neutrophil presence in the lung (data not shown), and by total lung neutrophil count after enzymatic digestion, with maximal levels noted from 2 to 7 days after bead administration (Figure 1B). Importantly, the influx of neutrophils was temporally associated with an induction of TNF-{alpha} and glutamine-leucine-arginine (ELR) + CXC chemokines CXCL5/lipopolysaccharide-induced CXC chemokine, KC, and MIP-2, with maximal expression observed at 2 to 3 days after bead administration (Figure 1C).



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  		Figure 1. Effect of intratracheal administration of 5 x 105 cfu of Pseudomonas beads on lung bacterial load (A), lung neutrophil number (B), and tumor necrosis factor-{alpha} (TNF-{alpha}), and glutamine-leucine-arginine (ELR) + cysteine-X-cysteine (CXC) chemokines, macrophage inflammatory protein-2 (MIP-2), CXCL5/lipopolysaccharide-induced CXC chemokine (LIX), and keratinocyte-derived chemokine (KC) levels (C) at Days 1, 2, 7, and 12 after Pseudomonas beads challenge (experimental n = 5 animals per group per time point). A depicts log cfu/ml of Pseudomonas. B depicts total lung neutrophil count (x106 cells/lung). C depicts fold induction of TNF-{alpha}, MIP-2, CXCL5/lipopolysaccharide-induced CXC chemokine, and KC in whole lung homogenates above levels in sterile beads-treated animals. All data were expressed as mean ± SEM. *p < 0.01 for infected mice compared with mice administered sterile beads.

 
Effect of Azithromycin Treatment on Lung Bacterial Clearance
The serum and lung concentrations of azithromycin dose used in this study mimicked levels that are routinely achieved with normal dosing of this drug in humans (as discussed in METHODS) and were well below the typical minimal inhibitory concentrations (MICs) (> 8 µg/ml at 18 hours) for our mucoid Pseudomonas strain. To determine whether this noninhibitory concentration of azithromycin would affect pulmonary bacterial clearance, mice were subcutaneously administered azithromycin (or received no treatment) and then infected with Pseudomonas beads. Lungs were harvested 1, 3, and 6 days after intratracheal inoculation. Animals administered azithromycin had no difference in the number of mucoid P. aeruginosa cfu isolated from the lung, as compared with control untreated animals challenged with Pseudomonas beads (Figure 2). These results indicate that treatment with azithromycin at concentrations that are routinely achieved with normal dosing of this drug and well below the MIC of our mucoid P. aeruginosa did not alter pulmonary clearance of the bacteria.



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  		Figure 2. Effect of azithromycin on lung bacterial clearance 1, 3, and 6 days after intratracheal administration of 5 x 105 cfu of Pseudomonas beads (experimental n = 4 animals per group). Data are representative of three separate experiments. Azi = azithromycin 20 mg/kg per mouse subcutaneously once daily for 3 days. Control animals received no treatment.

 
Effect of Azithromycin on Lung Inflammatory Cell Influx after Intratracheal Inoculation with Pseudomonas Beads
To determine whether the administration of azithromycin alters the recruitment of inflammatory cells to the airways, azithromycin-treated animals or untreated control animals were challenged with mPa beads, and then lungs were harvested at 3 days and enzymatically digested for cell count enumeration. This time point was chosen because maximum influx of neutrophils in response to the intratracheal administration of Pseudomonas beads occurs between 2 to 7 days after mPa challenge. The administration of azithromycin resulted in a marked decrease in the total number of cells, which was largely due to a substantial reduction in neutrophils (p < 0.05) (Figure 3A). Treatment with azithromycin did not significantly alter the numbers of macrophages or lymphocytes (data not shown). Histopathologic evaluation 2 days after P. aeruginosa–impregnated bead administration showed an infiltration of predominantly neutrophils in a patchy, endobronchial, and peribronchial pattern corresponding to areas of bead deposition. After azithromycin treatment, decreased inflammation with dramatically less cellular infiltrates and a paucity of neutrophils was evident (Figure 3B). These results indicate that treatment with azithromycin resulted in impaired recruitment of neutrophils into the lungs during endobronchial infection by P. aeruginosa.




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  		Figure 3. (A) Effect of azithromycin on lung cellular influx 3 days after intratracheal administration of 5 x 105 cfu of Pseudomonas (mPa) beads (experimental n = 5 animals per group). Uninfected = mice without mPa beads or azithromycin treatment; mPa5 = mice after intratracheal bead mPa beads but no azithromycin treatment; mPa5 +Azi= mice after intratracheal mPa beads and azithromycin 20 mg/kg per mouse subcutaneously once daily for 3 days. *p < 0.01 for infected, untreated mice compared with infected, azithromycin-treated mice. Data are representative of three separate experiments. (B) Effect of azithromycin on lung histopathology 3 days after intratracheal administration of 5 x 105 cfu of mPa beads (experimental n = 2 animals per group [magnification x100]). Control depicts representative lung hematoxylin and eosin stains in mice receiving saline treatment; azithromycin depicts representative lung hematoxylin and eosin stains in mice receiving azithromycin at 20-mg/kg per mouse subcutaneously once daily for 3 days.

 
Effect of Azithromycin Administration on the Production of Cytokines within the Lung after Intratracheal Challenge with Pseudomonas Beads
Subsequent experiments were performed to determine whether the azithromycin-dependent reduction of neutrophil recruitment resulted from altered production of important proinflammatory cytokines. Several cytokines have been shown to be crucial to antibacterial host defense in Gram-negative infection, including TNF-{alpha}, T1-phenotype cytokines (IFN-{gamma}, IL-12), and specific members of the ELR + CXC chemokine family (MIP-2, KC, and CXCL5/lipopolysaccharide-induced CXC chemokine), which preferentially recruit and activate neutrophils. The baseline levels of these cytokines were low or not detectable in lungs of uninfected control or azithromycin-treated animals (data not shown). The intratracheal administration of mPa beads resulted in maximal expression of cytokines at 2 to 3 days in both azithromycin-treated and control infected mice, representing at least a twofold increase in protein levels of all cytokines assessed (TNF-{alpha}, IFN-{gamma}, IL-12, MIP-2, KC, and lipopolysaccharide-induced CXC chemokine) when compared with that seen in lung homogenates prepared from animals administered sterile beads (Figure 1C). There was a significant reduction in lung levels of TNF-{alpha} in azithromycin-treated infected mice as compared with control infected mice (Figure 4A). Furthermore, a significant decrease in lung KC levels was observed in azithromycin-treated infected animals (Figure 4B), whereas MIP-2 and lipopolysaccharide-induced CXC chemokine levels were not altered by azithromycin administration (data not shown). Levels of IL-12 and IFN-{gamma} were not different in azithromycin-treated and control groups (data not shown). These results indicate that azithromycin treatment is associated with the selective reduction of lung TNF-{alpha} and KC levels.



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  		Figure 4. Effect of azithromycin on lung TNF-{alpha} (A) and KC (B) levels 3 days after intratracheal administration of 5 x 105 cfu of mPa beads (experimental n = 8 animals per group). Uninfected = mice without mPa beads or azithromycin treatment; Sterile beads = mice after intratracheal sterile beads and no azithromycin treatment; mPa5 control = mice after intratracheal mPa beads but no azithromycin treatment; mPa5 + Azi = mice after intratracheal mPa beads and azithromycin treatment. Azithromycin treatment consists of 20 mg/kg per mouse subcutaneously for 3 days. *p < 0.05; **p < 0.02 for infected, untreated mice compared with infected, azithromycin-treated mice. Data are representative of three separate experiments. All data are expressed as mean cytokine concentration (ng/ml) ± SEM.

 
Effect of Azithromycin on Neutrophil Chemotactic Responses In Vitro
Because azithromycin appears to modulate neutrophil recruitment to a greater degree than the more modest change in inflammatory cytokines (25–35% reduction), we questioned whether azithromycin had direct effects on the ability of neutrophils to migrate in response to chemotactic stimuli. We had initially confirmed that incubation of neutrophils with azithromycin was not associated with loss of cell viability or toxicity (see METHODS). Interestingly, migration of peritoneal-elicited mouse neutrophils in response to either FMLP or KC was significantly reduced when cells were preincubated in vitro with azithromycin for 4 hours (Figure 5A). The ability of azithromycin-treated mouse neutrophils to migrate in response to MIP-2 was also similarly reduced (data not shown). Furthermore, azithromycin treatment produced a similar and impressive attenuation of chemotactic response to FMLP and IL-8 in human neutrophils (Figure 5B). The results indicate that azithromycin directly reduced neutrophil chemotaxis in response to chemotactic stimuli. In addition, azithromycin appears to regulate both chemokine-dependent and chemokine-independent neutrophil chemotactic responses.



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  		Figure 5. Effect of 4-hour preincubation of 4-µg/ml azithromycin (open bars) or control Hanks' balanced salt solution (HBSS) (solid bars) on murine neutrophil chemotaxis in response to HBSS, N-formyl-met-leu-phe (FMLP; 10–7 M), and KC (20 ng/ml) (A), and on human neutrophil chemotaxis in response to HBSS, FMLP, and interleukin (IL)-8 (1 ng/ml) (B). *p < 0.01; **p < 0.001 for azithromycin-treated cells compared with untreated cells. Data are representative of four separate experiments each. All data are expressed as mean neutrophil count per high-powered field at x1,000 magnification (neutrophils per high-powered field) ± SEM.

 
Effect of Clarithromycin on Neutrophil Chemotactic Responses In Vitro
To determine whether the observed effects on chemotaxis in our experimental model in vitro are shared by other members of the macrolide family or are unique to azithromycin, we chose to test whether clarithromycin, a 14-membered ring macrolide, had a similar effect on the ability of neutrophils to migrate in response to chemotactic stimuli. Clarithromycin treatment produced a similar and significant inhibition of human neutrophil chemotaxis in response to IL-8 in human neutrophils. Clarithromycin-treated cells demonstrated significantly more neutrophil chemotactic inhibition when compared with azithromycin-treated cells after FMLP stimulation. These comparison studies suggest that the immunosuppressive effects are not specific for azithromycin and may involve structurally different members of the macrolide family (Figure 6).



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  		Figure 6. Effect of 4-hour preincubation of control HBSS, 4 µg/ml of azithromycin, or 10 µg/ml of clarithromycin on murine neutrophil chemotaxis in response to HBSS, FMLP, and IL-8. *p < 0.001 for macrolide-treated cells compared with untreated cells. Data are representative of three separate experiments each. All data are expressed as mean neutrophil count per high-powered field at x1,000 magnification (neutrophils per high-powered field) ± SEM.

 
Effect of Azithromycin on Activation of PI3K and MAPK Signaling Pathways in Neutrophils after Exposure to Chemotaxins
The observation that azithromycin reduced neutrophil recruitment by divergent chemoattractants FMLP and chemokines KC or IL-8 suggests a potential common regulatory mechanism by which azithromycin affects neutrophils. Both FMLP and IL-8 are known to activate multiple effector pathways after binding to respective cell surface G-protein–coupled receptors on neutrophils. Of interest, chemotactic responses induced by IL-8 and FMLP stimulation involve activation of the PI3K pathway and MAPK pathways, specifically the p38 MAPK and p42/44 (extracellular signal-related kinase [ERK]-1/2) MAPK cascades (41–44). Neutrophil chemotactic responses to FMLP have been reported to be dependent on p38 MAPK, whereas IL-8–induced migration of neutrophils favors the PI3K/AKT pathway (45). However, growing evidence indicates that ERK-1/2 may play an important role in neutrophil migration in response to FMLP (44, 46–48) or IL-8 (49–52). We therefore performed experiments to determine the relative role of specific signal transduction pathways in regulating chemotactic responses to either FMLP or IL-8 using chemical inhibitors of selective PI3K and MAPK pathways. Importantly, FMLP-stimulated chemotaxis of human neutrophils was significantly reduced by U0126, a specific inhibitor of MAP kinase ERK kinase 1 (MEK1)/ERK-1/2, but was not significantly altered by LY294002 (a PI3K inhibitor) or the p38 MAPK inhibitor SB20358 (Figure 7). Likewise, inhibition of ERK-1/2 blocked IL-8–induced neutrophil chemotaxis, whereas preincubation of cells with either LY294002 or SB20358 had no significant effect on neutrophil chemotaxis to IL-8 (data not shown). In dose–response studies, concentrations from 1 to 30 µM of LY294002 (PI3K inhibitor) and SB203580 (p38 MAPK inhibitor) did not have greater inhibitory effect on FMLP-induced chemotaxis. In our studies, PI3K did not appear to play a major role in neutrophil recruitment.



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  		Figure 7. Effect of pharmacologic inhibition of p38 mitogen-activated protein kinase (MAPK) (SB20358), extracellular signal-related kinase (ERK)-1/2 (U0126), and phosphatidylinositol 3-kinase (PI3K) (LY294002) on human neutrophil chemotaxis in response to FMLP (10–7 M). Dimethyl sulfoxide is used as a diluent for chemical inhibitors and represents uninhibited control. *p < 0.01 for inhibitor-treated cells compared with untreated cells. Data are sum of three separate experiments each. All data are expressed as mean neutrophil count per high-powered field (hpf) at x1,000 magnification (neutrophils per hpf) ± SEM.

 
Because the most profound effect of azithromycin was observed with ERK-1/2 inhibition, we next determined whether this macrolide influenced the activation of ERK-1/2 in neutrophils on exposure to both IL-8 and FMLP chemoattractants. We observed a greater magnitude of MAPK activation in response to FMLP, as compared with stimulation with IL-8. After stimulation with FMLP, there was an impressive time-dependent increase in the accumulation of phosphorylated ERK-1/2 (as a surrogate indicator of ERK-1/2 activation), which was maximal at 2 minutes after stimulation but persisted to 60 minutes after FMLP. Interestingly, pretreatment of the neutrophils with azithromycin (4 µg/ml) inhibited FMLP-induced ERK-1/2 activation early at 2 and 10 minutes but dramatically reduced ERK-1/2 activation by nearly 90% at later time points of 30 and 60 minutes, as compared with neutrophils not incubated with azithromycin (Figures 8A and 8C). Furthermore, treatment of human neutrophils with azithromycin resulted in a more rapid decrease in phosphorylated ERK-1/2 levels relative to that observed in untreated cells. In contrast, pretreatment of neutrophils with azithromycin caused minimal changes in the FMLP-induced activation of p38 (data not shown), suggesting some selectivity in the inhibitory effects of azithromycin. There was also a time-dependent increase in the accumulation of phosphorylated ERK-1/2 in response to IL-8, which was also maximal at 2 minutes after stimulation and persisted to 30 minutes after IL-8. Similarly, pretreatment of the neutrophils with azithromycin inhibited IL-8–induced ERK-1/2 activation at 2 and 10 minutes but modestly at 30 minutes as compared with neutrophils not incubated with azithromycin (Figure 8B and D). For all time points, the ERK-1 (44) band is noted to be preferentially phosphorylated compared with the ERK-2 (42) band after stimulation with both FMLP and IL-8. We therefore selected phosphorylated ERK-1 (p-ERK-1) and total ERK-1 for densitometry quantitation. The densitometry for p-ERK-1 was normalized to the total ERK-1 and shown as fold increase in band density above that of Hanks' balanced salt solution control. Collectively, these findings suggest that azithromycin may block neutrophil chemotaxis through both chemokine-dependent and -independent means by negatively regulating activity through the ERK-1/2 pathway.



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  		Figure 8. Effect of azithromycin on phosphorylation of ERK-1/2 in neutrophils after FMLP (A) or IL-8 (B) stimulation. Human neutrophils (5 x 106 per condition) were preincubated with (+) or without (–) azithromycin and stimulated with 10–7 M of FMLP (A) or with 100 ng/ml of IL-8 (B) at 37°C for 2, 10, 30, and 60 minutes. Unstimulated control neutrophils in HBSS were evaluated at 0 minute. The cells were then washed and lysed. Western blots were probed with an anti–phospho-specific antibody to ERK-1/2 and reprobed with an antibody to total ERK-1/2. Blots shown are representative of three separate experiments. Densitometry of blots depicted in A and B, expressed as fold increase of ERK-1 arbitrary intensity units above that of HBSS control, normalized to total ERK-1 to correct for loading differences (C and D). Azithromycin = neutrophils preincubated with azithromycin and stimulated with FMLP (C) or IL-8 (D); untreated = neutrophils preincubated without azithromycin and stimulated with FMLP (C) or IL-8 (D). Densitometries shown are representative of three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We chose a model of murine endobronchial mPa infection in our studies because it most closely mimicked the chronic bacterial colonization seen in human cystic fibrosis airway disease. Azithromycin was selected as a potential immunomodulatory macrolide because of a growing number of reports suggesting beneficial effects of this macrolides in both diffuse panbronchiolitis and cystic fibrosis (10, 13, 14, 33, 53, 54), diseases characterized by the persistence of P. aeruginosa within the airway and progressive airway destruction. Results recently reported from a multicenter trial in patients with cystic fibrosis treated with low-dose azithromycin suggest significant improvements in clinical outcomes (16). However, the mechanism(s) by which azithromycin affords these beneficial effects was not investigated. In our model, azithromycin treatment in vivo resulted in an impressive antiinflammatory effect, manifested primarily by attenuation of TNF-{alpha} and KC expression and inhibition of neutrophil chemotactic response, effects that occurred without changes in lung bacterial burden. Our study confirms the previously demonstrated lack of bactericidal effect on P. aeruginosa at physiologically achievable doses in vivo. We did not ascertain the degree of suppression of virulence factors in our mucoid Pseudomonas strain but acknowledge the strong possibility that bacterial pathogenicity may be partially impaired at the concentration of azithromycin achieved in the in vivo studies (55–57). Inhibition of mPa virulence may be an important mechanism by which azithromycin affects the host inflammatory response. However, establishing the interaction of azithromycin-affected bacteria with the host immune response is beyond the scope of this study. Our observation of a direct azithromycin effect on neutrophil recruitment is in keeping with the suggestion that macrolides also directly target multiple components of the inflammatory cascade in the absence of bacterial effect (22, 26). Because the enhanced intracellular drug accumulation in host phagocytes may be a major regulatory mechanism of azithromycin effects on the host response, we investigated the cellular response in vivo after treatment with azithromycin. The reduction in neutrophil influx after azithromycin treatment supported clinical observations made in patients with diffuse pan-bronchiolitis treated with erythromycin (10, 54).

In our study, we found a significant reduction in the in vivo production of proinflammatory cytokines TNF-{alpha} and the ELR + CXC chemokine KC in the lungs of azithromycin-treated mice. No differences in levels of IL-12, IFN-{gamma}, or other CXC chemokine family members tested were noted, demonstrating some selectivity in suppressive effects. The cytokine-producing cells, which are directly influenced by azithromycin remain unknown but do not appear to be the alveolar macrophages, as the in vitro incubation of primary mouse alveolar macrophages with azithromycin failed to alter the LPS-induced production of TNF-{alpha}, KC, or IL-12 from these cells (data not shown). It is quite possible that the airway epithelium remains an important source of cytokines in airway-localized infection, and these cells may represent a cellular target of azithromycin. Consistent with this notion, macrolides have previously been shown to inhibit the expression of inflammatory cytokines from airway epithelial cells in vitro, an effect that is mediated by suppression of nuclear factor-{kappa}B and activator protein-1 (AP-1). It is also plausible that the reduction in lung TNF-{alpha} and KC may be due to the overall decreased number of cytokine-producing host immune cells (e.g., neutrophils) recruited into the lung after azithromycin treatment. Although the diminished levels of TNF-{alpha} supports earlier reports of antiinflammatory effect of macrolides (17, 32, 58, 59), we found no effect of the azithromycin on the expression of other proinflammatory cytokines previously found to be regulated by various macrolides antibiotics. Azithromycin treatment also appears to cause selective reduction of CXC chemokines known to recruit neutrophils, as only KC was significantly diminished. This reduction in KC may be sufficient to affect negatively neutrophil recruitment to the lungs, despite the fact that other members of the CXC chemokine family were not altered by macrolide treatment. However, it is more likely that azithromycin effects are only partially mediated by suppression of activating and chemotactic cytokines in our chronic endobronchial infection model.

We further observed a substantial impairment in the ability of neutrophils to migrate in response to FMLP and ELR + CXC chemokines in vitro after treatment with azithromycin. The evidence for a direct effect of macrolides on neutrophil chemotaxis is conflicting. Some studies indicated that treatment with macrolides, including erythromycin, josamycin, and roxithromycin, might actually enhance neutrophil migration in response to chemoattractants (23, 26). In contrast, others have shown that in vitro incubation of neutrophils with selected macrolides (erythromycin, josamycin, miokamycin, roxithromycin, or rokitamycin) significantly impaired the migration of neutrophils (60). The reasons for this disparity in results are uncertain but may relate to differences in doses of macrolides employed, duration of exposure, or activational state of the neutrophils studied. We observed a similar and significant inhibition of neutrophil chemotaxis by clarithromycin, suggesting that the immunosuppressive effects are not specific for azithromycin. We speculated that azithromycin might directly affect synthesis of bacterial protein and other virulent factors, without affecting bacterial viability, enough to diminish the bacteria's ability to stimulate neutrophils in our in vivo model. However, our in vitro studies suggest that azithromycin can directly and substantially attenuate neutrophil recruitment in the absence of whole bacteria. These effects may involve structurally different members of the macrolide family.

Our observation that azithromycin can result in an abrogation of neutrophil responses from distinctly different stimuli suggests that azithromycin likely regulates chemotaxis through common or combinatorial downstream intracellular pathways rather than altering ligand binding or receptor expression. Consistent with this notion, azithromycin reduced activation of the ERK-1/2 MAPK signal transduction pathway, a pathway that is known to be induced by both IL-8 and FMLP (41–44). Limited data in neutrophils and other cell systems suggest that macrolides might exert their immunomodulatory effects by targeting intracellular signaling pathways. For example, the uptake of macrolides involves the p38 MAP kinase (61), whereas the L-cladinose ring, characteristic of all erythromycin A–derived macrolides, impairs oxidant production by interfering with the phospholipase D–phosphatidate phosphohydrolase pathways (62). Recently, clarithromycin has been noted to inhibit overproduction of the muc5ac core protein in a murine model of diffuse pan-bronchiolitis through modulation of ERK-1/2 activation (63). Another possible mechanism by which azithromycin might influence neutrophil mobility is by altering intracellular calcium flux, a process that is require for chemotactic responses (64). Whether there is a unifying mechanism by which azithromycin affects receptor or postreceptor transductional events involved in the activation of chemotactic pathway will be the subject of further investigation.

Cystic fibrosis remains a devastating disease because of persistent airway colonization by Pseudomonas resulting in chronic unopposed neutrophil-dominated airways inflammation (3). Macrolides such as azithromycin may be important contributors to the cystic fibrosis antiinflammatory armamentarium by reducing local tissue injury. There remains concern that attempts at inhibiting a vigorous and early host response required to clear pathogenic bacteria effectively from the airway may have detrimental effects (19, 37, 38, 65–67). Importantly, we did not observe a negative effect on host defense, as animals treated with azithromycin did not have an increased bacterial burden despite the reduction in inflammatory cell recruitment. The precise mechanisms by which azithromycin attenuate cellular host response in our endobronchial Pseudomonas infection model warrant further investigation. Our observations provide mechanistic support for the use of chronic azithromycin therapy as a complementary therapeutic strategy to be employed in inflammatory airways disease.


   Acknowledgments
 
The authors gratefully acknowledge Dr. Dennis Girard (Global Research & Development Division, Pfizer, Inc., Groton, CT) for making the azithromycin pharmacokinetic bioassay determinations.


   FOOTNOTES
 
Supported in part by National Institutes of Health grants KO8HL04421 and 1 HD28820.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: W.C.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.L.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.S.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.C.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; V.J.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.B.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; T.J.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 17, 2004; accepted in final form September 7, 2004


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 METHODS
 RESULTS
 DISCUSSION
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